This application addresses broad Challenge Area (11) Regenerative Medicine and specific Challenge Topic 11-AR-101: Musculoskeletal and Skin Tissue Regeneration. The focus of this grant is to develop new strategies for muscular dystrophy therapies or/and strengthen the existing strategies. This grant is based on a new finding showing that reduction of LPP3 that reduces the level of active second messenger, S1P can suppress dystrophic phenotypes in Drosophila melanogaster. We will now study the mechanism of sphingolipid pathway and Dystrophin interactions and further, will test for wunen/LPP3 based strategies to ameliorate muscle function in dystrophic mice. Muscular Dystrophy is a muscle wasting disease that at present has no cure. As mentioned, our studies have produced a possible new inroad into treatment for this disease and it involves wunen. We will exhaustively characterize wunen, a suppressor isolated in our previous forward genetic screens for modifiers of the Dystrophin-associated Glycoprotein Complex mutant phenotypes. Wunen is a homolog of human This grant is based on a new finding showing that reduction of LPP3 that reduces the level of active second messenger, S1P can suppress dystrophic phenotypes in Drosophila melanogaster. We will now study the mechanism of sphingolipid pathway and Dystrophin interactions and further, will test for wunen/LPP3 based strategies to ameliorate muscle function in dystrophic mice. Muscular Dystrophy is a muscle wasting disease that at present has no cure. As mentioned, our studies have produced a possible new inroad into treatment for this disease and it involves wunen (LPP3) which many studies have shown is involved in the regulation of levels of bioactive lipids, particularly sphingosine 1-phosphate (S1P). S1P is implicated in pre-myoblast (adult muscle stem cell) maintenance as well as myoblast differentiation into myotubes. The possibility of a muscle regenerative strategy for suppressing the muscular dystrophy phenotype through the alteration of sphingolipid signaling has high potential for being extremely fruitful. We will determine if wunen acts through S1P to inhibit apoptosis and/or increase muscle generation through either increasing proliferation of adult muscle precursor cells or increasing proliferation of adult muscle satellite stem cells which have yet to be defined in Drosophila. We will determine if muscle satellite stem cells exist in the same physical niche as mammalian muscle satellite stem cells which is between the basal lamina and the sarcolemma of existing muscle fibers. In toto, we will establish and extend the role of sphingolipid signaling in muscle formation in Drosophila and determine the mechanism that alleviates the disease phenotype of dystrophic flies. Furthermore, we will test whether reduction of Wunen/LPP3 can be advantageous in muscular dystrophy to mammalian animal models of the disease, particularly the mdx mouse (and in the future the dog model). We hope to produce regenerative therapeutic strategies to alleviate the disease phenotype in this model. These strategies will include reducing LPP3 function as well as increasing the presence of its downstream effector S1P. We propose that increasing sphingolipid signaling will increase the regeneration potential of muscle stem cells and myoblasts and thereby will improve the efficacy of muscular dystrophy therapy. We will test this hypothesis by two different approaches. First we will test whether reduced LPP3 and/or increased S1P levels will increase muscle cell regeneration in mdx mice using the existing muscle regeneration methods in Seattle Muscular Dystrophy Consortium (2.1). Second, we will take advantage of the induced pluripotent stem cell (iPSC) expertise in my laboratory to generate skeletal myoblasts using iPSCs derived from muscular dystrophy patients and test whether the regeneration capacity of these myoblasts is increased due to the modified levels of LPP3 and/or S1P (2.2). This challenge grant combines multiple fields;two Seattle based consortiums and three laboratories. The work from this combination of disciplines is bound to generate new exciting advances in muscular dystrophy therapy The goal of this grant is to generate a potential therapy for muscular dystrophy using a lipid phosphate phosphatase, Wunen/LPP3. Reduction of this particular lipid phosphatase shows partial rescue of defects seen in a Drosophila muscular dystrophy model. We will now test whether reduction of this phosphatase, LPP3 can suppress muscle defects in multiple muscular dystrophy model systems. Ultimately, in the future we wish to test whether manipulating sphingolipid metabolism can serve therapeutic function in muscular dystrophy treatment.